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Purity: ≥98%
Description: BI-1347 is a novel, potent and selective CDK8 inhibitor with an IC50 of 1.1 nM. Compound I-003 from patent WO2017202719A1
| Targets |
CDK8 (IC50 = 1.1 nM)
CDK8 (IC50 = 1.4 nmol/L) and CDK19.[2] |
|---|---|
| ln Vitro |
BI-1347 (150 nM; 44 h) increases the production of granzyme B (GZMB+) in mouse splenic NK cells[2].
BI-1347 (0.1 nM-10 μM; 24 h) treatment causes NK92MI cells to secrete more perforin[2]. BI-1347 is a highly selective small molecule inhibitor of CDK8/19. It potently decreases phosphorylation of STAT1S727 in NK cells, which augments the production of cytolytic molecules perforin and granzyme B.[2] Treatment of the human NK cell line NK92MI with BI-1347 increased perforin secretion into the supernatant.[2] In murine splenocytes activated with IL-15, treatment with 150 nmol/L BI-1347 increased the proportion of granzyme B-positive NK cells by approximately 4-fold.[2] Treatment with BI-1347 did not affect the proliferation of the NK92MI cell line, nor did it affect CDK8 protein levels or phosphorylation of STAT1Tyr701 and STAT3Tyr705 in these cells.[2] In a panel of cancer cell lines, BI-1347 showed growth inhibition (IC50 < 1 µM) in only a small subset of hematological lines tested (e.g., MV-4-11B, KG-1). None of the solid cancer cell lines tested, including MC-38 and EMT6, were affected.[2] No change in viability was observed in primary human AML samples or primary human NK cells after incubation with BI-1347.[2] BI-1347 enhanced antibody-dependent cellular cytotoxicity (ADCC). The combination of BI-1347 with the CD20 antibody rituximab enhanced ADCC-mediated depletion of primary chronic lymphocytic leukemia (CLL) cells in the presence of PBMCs or purified NK cells. This enhanced effect was not observed in the absence of NK cells.[2] The CDK8/19 inhibitors (including BI-1347) displayed exquisite selectivity for CDK8/19 with a selectivity ratio of >300-fold towards all other kinases tested (out of 326 kinases). Only CDK19, the close homolog of CDK8, was equipotently inhibited.[2] |
| ln Vivo |
BI-1347 (oral gavage; 10 mg/kg; once daily; 30 d) exhibits anti-tumor activity and modulates STAT1S727 phosphorylation in vivo[2]. In the mammary carcinoma EMT6 model, the combination of BI-1347 intermittent schedule and BI-8382 continuous treatment increases efficacy compared to each monotherapy[2].
In the murine B16-F10-luc2 syngeneic melanoma model, daily oral administration of BI-1347 (10 mg/kg) resulted in significant tumor growth inhibition (TGI of 94% on day 23 and 88% on day 29) and a median survival advantage of 4 days compared to vehicle-treated animals.[2] This in vivo efficacy contrasted with the in vitro resistance of B16-F10 cells to CDK8/19 inhibitors, suggesting an immune-mediated mechanism.[2] In the murine MC-38 colon adenocarcinoma model, combination of BI-1347 (10 mg/kg, daily oral) with an anti-PD-1 antibody (10 mg/kg, i.p., twice weekly) led to significantly enhanced anti-tumor response (TGI = 97%) compared to either monotherapy.[2] In the murine EMT6 mammary carcinoma model, daily oral monotherapy with BI-1347 (10 mg/kg) did not significantly increase median survival. An intermittent schedule (5 days on / 5 days off) of BI-1347 resulted in a median survival of 26 days vs 22 days for control.[2] Combining the SMAC mimetic BI-8382 (50 mg/kg, daily) with intermittent BI-1347 (10 mg/kg, 5 days on/5 days off) synergistically increased efficacy, resulting in a median survival of 45 days and complete tumor regression in 5 out of 10 mice.[2] The survival benefit of the BI-1347 and SMAC mimetic combination was dependent on NK cells, as depletion of NK cells abolished the enhanced efficacy, reducing it to the level of SMAC mimetic monotherapy.[2] Depletion of CD8+ T cells in the combination group reduced efficacy to a level comparable to BI-1347 monotherapy.[2] A single oral dose of BI-1347 (3 mg/kg) significantly reduced STAT1S727 phosphorylation in murine splenocytes.[2] |
| Enzyme Assay |
A LanthaScreen Eu kinase binding assay was used to identify compounds that competitively interact with the ATP binding pocket of CDK8. The assay utilized CDK8/CycC complex, a biotinylated anti-His tag antibody, Eu-labeled streptavidin, and a kinase tracer. Compounds were tested for their ability to displace the tracer, indicating binding to the kinase's ATP site.[2]
Selectivity profiling was performed using a commercial kinase profiling service. Single-point measurements were conducted using 1 µM of test compound. For IC50 determinations, compounds were tested starting at 20 µM with subsequent 10-fold serial dilutions.[2] |
| Cell Assay |
Cell Line: Mouse splenic NK cells
Concentration: 150 nM Incubation Time: 44 hours Result: Increased the proportion of granzyme B-positive NK cells by approximately 4-fold. For viability assays, cells were plated in 96-well plates and treated with BI-1347 at various concentrations for 72-120 hours. Viability was assessed using luminescent or fluorescent cell viability assays. For primary AML and NK cells, viability was determined by flow cytometry after staining with annexin V and propidium iodide.[2] For the perforin release assay, NK92MI cells were seeded and treated with compounds for 24 hours. The amount of perforin secreted into the supernatant was quantified by ELISA.[2] For immunoblotting analysis of STAT1 phosphorylation, NK92MI cells or primary NK cells were treated with compounds for 6 hours, often followed by stimulation with cytokines like IFN-β or IL-2 for 1 hour. Cells were lysed and proteins were analyzed by Western blot using specific phospho-antibodies.[2] To assess granzyme B modulation in mouse splenocytes, cells were cultured with IL-15 and/or BI-1347 for 44 hours. A protein transport inhibitor was added for the last 4 hours. Cells were then analyzed by flow cytometry for intracellular granzyme B staining or by Western blot.[2] For ADCC assays using rituximab on primary CLL cells, leukemia cells were co-cultured with autologous PBMCs or purified NK cells in the presence of rituximab and BI-1347. Leukemia cell depletion or death was analyzed by flow cytometry after 48 hours or 4 hours, respectively.[2] |
| Animal Protocol |
B16-F10-luc2 syngeneic melanoma model[2]
10 mg/kg Oral gavage; 10 mg/kg; once daily; 30 d For efficacy studies in the B16-F10-luc2 melanoma model, female C57BL/6N mice were engrafted intravenously with tumor cells. Treatment with BI-1347 (10 mg/kg, daily oral) or vehicle was initiated after randomization based on body weight. Tumor growth was monitored by in vivo imaging.[2] For the MC-38 colon adenocarcinoma model, female C57BL/6N mice were engrafted subcutaneously. Mice were treated with BI-1347 (10 mg/kg, daily oral) and/or an anti-PD-1 antibody (10 mg/kg, intraperitoneal, twice weekly).[2] For the EMT6 breast cancer model, female BALB/c mice were engrafted subcutaneously. Mice were treated with BI-1347 (10 mg/kg, daily oral or 5 days on/5 days off oral), the SMAC mimetic BI-8382 (50 mg/kg, daily oral), or their combination. Tumor volume and survival were monitored.[2] For immune cell depletion studies, an anti-asialo-GM1 antibody (for NK cell depletion) or an anti-CD8 antibody (for CD8+ T cell depletion) was administered intraperitoneally according to specific schedules starting several days before BI-1347 treatment.[2] For pharmacokinetic and pharmacodynamic studies, C57BL/6 mice were dosed orally or intravenously with BI-1347. Plasma was collected at various time points for concentration analysis. For biomarker analysis, spleens were harvested at specified times after a single dose to analyze STAT1 phosphorylation in immune cells by flow cytometry or Western blot.[2] |
| ADME/Pharmacokinetics |
BI-1347 showed a low clearance rate in mice (14% of hepatic blood flow). [2] It exhibited excellent oral exposure and bioavailability (100% in mice). [2] After a single oral dose of 25 mg/kg in mice, the plasma concentration of BI-1347 24 hours later was approximately 100 times its biochemical IC50 value for CDK8, indicating that daily administration can achieve complete coverage of the target. [2] In vitro experiments showed that BI-1347 exhibited higher metabolic stability in human hepatocytes (predicted hepatic clearance of 17%) compared to the previous compound (compound 2, 75%). [2]
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| Toxicity/Toxicokinetics |
In various preclinical animal models, 14 consecutive days of daily CDK8/19 inhibitor treatment was associated with adverse effects on stem cell compartments (gut, bone, immune system). [2]
In the B16-F10-luc2 efficacy study, daily oral administration of 10 mg/kg of BI-1347 was well tolerated with minimal effect on body weight. [2] This study suggests that intermittent administration of BI-1347 may improve the therapeutic index by avoiding the toxicities associated with continuous daily administration. [2] |
| References | |
| Additional Infomation |
BI-1347 is a tool compound available through Boehringer Ingelheim’s open innovation platform. [2]
This study suggests that the in vivo antitumor efficacy of selective CDK8/19 inhibitors (such as BI-1347) is primarily mediated through tumor-derived mechanisms, particularly through activation of NK cell-mediated antitumor immunity, rather than through direct antiproliferative effects on most cancer cells. [2] The efficacy of BI-1347 in combination with SMAC mimics depends on an intermittent treatment regimen, presumably designed to circumvent the state of immune hyporesponsiveness caused by chronic NK cell activation. [2] BI-1347 represents a novel immunotherapy strategy that enhances the activity of innate immunity (NK cells), which can be used in combination with drugs that modulate the adaptive immune system (e.g., anti-PD-1, SMAC). [2] |
| Molecular Formula |
C22H20N4O
|
|---|---|
| Molecular Weight |
356.420404434204
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| Exact Mass |
356.16
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| Elemental Analysis |
C, 74.14; H, 5.66; N, 15.72; O, 4.49
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| CAS # |
2163056-91-3
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| Related CAS # |
2163056-91-3
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| PubChem CID |
132180482
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
27
|
| Complexity |
503
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
WULUGQONDYDNKY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H20N4O/c1-25(2)22(27)15-26-14-19(12-24-26)16-7-9-17(10-8-16)21-13-23-11-18-5-3-4-6-20(18)21/h3-14H,15H2,1-2H3
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| Chemical Name |
2-[4-(4-isoquinolin-4-ylphenyl)pyrazol-1-yl]-N,N-dimethylacetamide
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| Synonyms |
BI-1347; BI1347; BI 1347
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 71~125 mg/mL (199.2~350.7 mM)
Ethanol: ~11 mg/mL (~30.9 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.84 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.84 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8057 mL | 14.0284 mL | 28.0568 mL | |
| 5 mM | 0.5611 mL | 2.8057 mL | 5.6114 mL | |
| 10 mM | 0.2806 mL | 1.4028 mL | 2.8057 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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